Bradykinin-Potentiating Activity of a Gamma-Irradiated Bioactive Fraction Isolated from Scorpion (Leiurus quinquestriatus) Venom in Rats with Doxorubicin-Induced Acute Cardiotoxicity: Favorable Modulation of Oxidative Stress and Inflammatory, Fibrogenic and Apoptotic Pathways.

Although doxorubicin (Dox) is a backbone of chemotherapy, the search for an effective and safe therapy to revoke Dox-induced acute cardiotoxicity remains a critical matter in cardiology and oncology. The current study was the first to explore the probable protective effects of native and gamma-irradiated fractions with bradykinin-potentiating activity (BPA) isolated from scorpion (Leiurus quinquestriatus) venom against Dox-induced acute cardiotoxicity in rats. Native or irradiated fractions (1 µg/g) were administered intraperitoneally (i.p.) twice per week for 3 weeks, and Dox (15 mg/kg, i.p.) was administered on day 21 at 1 h after the last native or irradiated fraction treatment. Electrocardiographic (ECG) aberrations were ameliorated in the Dox-treated rats pretreated with the native fraction, and the irradiated fraction provided greater amelioration of ECG changes than that of the native fraction. The group pretreated with native protein with BPA also exhibited significant improvements in the levels of oxidative stress-related, inflammatory, angiogenic, fibrogenic, and apoptotic markers compared with those of the Dox group. Notably, the irradiated fraction restored these biomarkers to their normal levels. Additionally, the irradiated fraction ameliorated Dox-induced histological changes and alleviated the severity of cardiac injury to a greater extent than that of the native fraction. In conclusion, the gamma-irradiated detoxified fraction of scorpion venom elicited a better cardioprotective effect than that of the native fraction against Dox-induced acute cardiotoxicity in rats.

Effect of a bradykinin-potentiating factor isolated from scorpion venom (Leiurus quinquestriatus) on some blood indices and lipid profile in irradiated rats.

Bradykinin appears to be an important regulator of cardiovascular function. It is also being increasingly noted as a participant in actions of drugs that affect the liver, kidney, and circulation. In our previous studies, bradykinin-potentiating factor (BPF) isolated from scorpion venom (Leiurus quinquestriatus) has been shown to be protective against hepato- and nephrotoxicity as well as healing skin burns by reducing oxidative stress in hyperglycemic conditions. Therefore, we aim to evaluate the ability of BPF in treating irradiated rats. A group of rats was exposed to γ-irradiation and subsequently treated with BPF injections aiming to elucidate the possibility of BPF to rescue γ-irradiation harmful effects. As controls, we used γ-irradiation exposed, BPF-injected, and untreated rats. The data obtained showed that the irradiated animals suffered from marked changes of many important blood parameters including red blood cells, leukocytes, platelets, hemoglobin, packed cell volume, high-density cholesterol, total cholesterol, triglycerides, and low-density cholesterol. Interestingly, BPF was able to rescue the deleterious effects of irradiation in rats and normalized their blood parameters to the basal levels. We conclude that BPF could ameliorate irradiation damaging effects.

A new structurally atypical bradykinin-potentiating peptide isolated from Crotalus durissus cascavella venom (South American rattlesnake).

Venom glands of some snakes synthesize bradykinin-potentiating peptides (BPP's) which increase bradykinin-induced hypotensive effect and decrease angiotensin I vasopressor effect by angiotensin-converting enzyme (ACE) inhibition. The present study shows a new BPP (BPP-Cdc) isolated from Crotalus durissus cascavella venom: Pro-Asn-Leu-Pro-Asn-Tyr-Leu-Gly-Ile-Pro-Pro. Although BPP-Cdc presents the classical sequence IPP in the C-terminus, it has a completely atypical N-terminal sequence, which shows very low homology with all other BPPs isolated to date. The pharmacological effects of BPP-Cdc were compared to BBP9a from Bothrops jararaca and captopril. BPP-Cdc (1 µM) significantly increased BK-induced contractions (BK; 1 µM) on the guinea pig ileum by 267.8% and decreased angiotensin I-induced contractions (AngI; 10 nM) by 62.4% and these effects were not significantly different from those of BPP9a (1 µM) or captopril (200 nM). Experiments with 4-week hypertensive 2K-1C rats show that the vasopressor effect of AngI (10 ng) was decreased by 50 µg BPP-Cdc (69.7%), and this result was similar to that obtained with 50 µg BPP9a (69.8%). However, the action duration of BPP-Cdc (60 min) was 2 times greater than that of BPP-9a (30 min). On the other hand, the hypotensive effect of BK (250 ng) was significantly increased by 176.6% after BPP-Cdc (50 µg) administration, value 2.5 times greater than that obtained with BPP9a administered at the same doses (71.4%). In addition, the duration of the action of BPP-Cdc (120 min) was also at least 4 times greater than that of BPP-9a (30 min). Taken together, these results suggest that BPP-Cdc presents more selective action on arterial blood system than BPP9a. Besides the inhibition of ACE, it may present other mechanisms of action yet to be elucidated.

Short peptide constructs mimic agonist sites of AT(1)R and BK receptors.

Extracellular peptide ligand binding sites, which bind the N-termini of angiotensin II (AngII) and bradykinin (BK) peptides, are located on the N-terminal and extracellular loop 3 regions of the AT(1)R and BKRB(1) or BKRB(2) G-protein-coupled receptors (GPCRs). Here we synthesized peptides P15 and P13 corresponding to these receptor fragments and showed that only constructs in which these peptides were linked by S-S bond, and cyclized by closing the gap between them, could bind agonists. The formation of construct-agonist complexes was revealed by electron paramagnetic resonance spectra and fluorescence measurements of spin labeled biologically active analogs of AngII and BK (Toac(1)-AngII and Toac(0)-BK), where Toac is the amino acid-type paramagnetic and fluorescence quencher 2, 2, 6, 6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid. The inactive derivatives Toac(3)-AngII and Toac(3)-BK were used as controls. The interactions characterized by a significant immobilization of Toac and quenching of fluorescence in complexes between agonists and cyclic constructs were specific for each system of peptide-receptor construct assayed since no crossed reactions or reaction with inactive peptides could be detected. Similarities among AT, BKR, and chemokine receptors were identified, thus resulting in a configuration for AT(1)R and BKRB cyclic constructs based on the structure of the CXCR(4), an α-chemokine GPCR-type receptor.

Inhibitory role of kinins on microglial nitric oxide and tumor necrosis factor-α production.

Brain inflammation is sustained by chronic activation of microglia and the over-production of pro-inflammatory cytokines and nitric oxide (NO), which in turn can be highly neurotoxic. Microglial activation can be regulated by neuropeptides such as bradykinin (BK) and other members of the kinin family. Kinins are well known inflammatory regulators outside the CNS. Although the kinin system is well distributed throughout the brain, the precise role of BK in the CNS is not yet clear. The aim of this study was to examine and characterize the effects of BK and related kinins on the production of NO and TNF-α in microglia. We found that BK and selective agonists for both B1 and B2 receptors, attenuated both NO and TNF-α levels in the media of BV2 microglial cells that had been stimulated with LPS. The effects of BK that were observed in BV2 cells were confirmed in primary neonatal rat microglial cells as well. In addition, all kinin agonists reduced the expression of iNOS and TNF-α protein and mRNA levels in LPS-stimulated BV2 cells. Also, while LPS activated the nuclear factor-κB (NF-κB) pathway, BK inhibited NF-κB activation by preventing degradation of the κB protein (IκB) inhibitor, abolishing translocation of p65 and p50 subunits to the nucleus and inhibiting NF-κB transcription activity. These results suggest a role for bradykinin in modulation of glial inflammation, as evidenced by attenuation of NO and TNF-α synthesis pathways in activated microglial cells.

Ensemble QSAR: a QSAR method based on conformational ensembles and metric descriptors.

Quantitative structure-activity relationship (QSAR) is the most versatile tool in computer-assisted molecular design. One conceptual drawback seen in QSAR approaches is the "one chemical-one structure-one parameter value" dogma where the model development is based on physicochemical description for a single molecular conformation, while ignoring the rest of the conformational space. It is well known that molecules have several low-energy conformations populated at physiological temperature, and each conformer makes a significant impact on associated properties such as biological activity. At the level of molecular interaction, the dynamics around the molecular structure is of prime essence rather than the average structure. As a step toward understanding the role of these discrete microscopic states in biological activity, we have put together a theoretically rigorous and computationally tractable formalism coined as eQSAR. In this approach, the biological activity is modeled as a function of physicochemical description for a selected set of low-energy conformers, rather than that's for a single lowest energy conformation. Eigenvalues derived from the "Physicochemical property integrated distance matrices" (PD-matrices) that encompass both 3D structure and physicochemical properties, have been used as descriptors; is a novel addition. eQSAR is validated on three peptide datasets and explicitly elaborated for bradykinin-potentiating peptides. The conformational ensembles were generated by a simple molecular dynamics and consensus dynamics approaches. The eQSAR models are statistically significant and possess the ability to select the most biologically relevant conformation(s) with the relevant physicochemical attributes that have the greatest meaning for description of the biological activity.

Lipopolysaccharides enhance the action of bradykinin in enteric neurons via secretion of interleukin-1beta from enteric glial cells.

Functional changes of the enteric nervous system have been observed under inflammatory states of inflammatory bowel disease increasing the endotoxin level. The aim of the present study was to determine the effect of lipopolysaccharides (LPS) on myenteric neuron-glia interaction in vitro. We examined the increase of the intracellular Ca(2+) concentration ([Ca(2+)](i)) and the release of interleukin-1beta (IL-1beta) or prostaglandin E(2) (PGE(2)) and COX-2 expression in myenteric plexus cells from the rat intestine induced by LPS. LPS potentiated BK-induced [Ca(2+)](i) increases in both myenteric neurons and enteric glial cells, which were suppressed by a B1R antagonist. Only in enteric glial cells, a B1R agonist increased [Ca(2+)](i). The effects of LPS were blocked by pretreatment with an interleukin-1 receptor antagonist or by reducing the density of enteric glial cells in culture. LPS prompted the release of IL-1beta from enteric glial cells. The augmenting effects of IL-1beta on the BK-induced neural [Ca(2+)](i) increase and PGE(2) release from enteric glial cells were abolished by a phospholipase A(2) (PLA(2)) inhibitor and a COX inhibitor, and partly suppressed by a COX-2 inhibitor. IL-1beta up-regulated the COX-2 expression in enteric glial cells. LPS promotes IL-1beta secretion from enteric glial cells, resulting in augmentation of the neural response to BK through PGE(2) release via glial PLA(2) and COX-2. The alteration of the regulatory effect of glial cells may be the cause of the changes in neural function in the enteric nervous system in inflammatory bowel disease.

Functional expression of kinin B1 and B2 receptors in mouse abdominal aorta

Previous studies have shown that the vascular reactivity of the mouse aorta differs substantially from that of the rat aorta in response to several agonists such as angiotensin II, endothelin-1 and isoproterenol. However, no information is available about the agonists bradykinin (BK) and DesArg9BK (DBK). Our aim was to determine the potential expression of kinin B1 and B2 receptors in the abdominal mouse aorta isolated from C57BL/6 mice. Contraction and relaxation responses to BK and DBK were investigated using isometric recordings. The kinins were unable to induce relaxation but concentration-contraction response curves were obtained by applying increasing concentrations of the agonists BK and DBK. These effects were blocked by the antagonists Icatibant and R-715, respectively. The potency (pD2) calculated from the curves was 7.0 ± 0.1 for BK and 7.3 ± 0.2 for DBK. The efficacy was 51 ± 2 percent for BK and 30 ± 1 percent for DBK when compared to 1 æM norepinephrine. The concentration-dependent responses of BK and DBK were markedly inhibited by the arachidonic acid inhibitor indomethacin (1 æM), suggesting a mediation by the cyclooxygenase pathway. These contractile responses were not potentiated in the presence of the NOS inhibitor L-NAME (1 mM) or endothelium-denuded aorta, indicating that the NO pathway is not involved. We conclude that the mouse aorta constitutively contains B1 and B2 subtypes of kinin receptors and that stimulation with BK and DBK induces contractile effect mediated by endothelium-independent vasoconstrictor prostanoids.

Functional expression of kinin B1 and B2 receptors in mouse abdominal aorta.

Previous studies have shown that the vascular reactivity of the mouse aorta differs substantially from that of the rat aorta in response to several agonists such as angiotensin II, endothelin-1 and isoproterenol. However, no information is available about the agonists bradykinin (BK) and DesArg(9)BK (DBK). Our aim was to determine the potential expression of kinin B(1) and B(2) receptors in the abdominal mouse aorta isolated from C57BL/6 mice. Contraction and relaxation responses to BK and DBK were investigated using isometric recordings. The kinins were unable to induce relaxation but concentration-contraction response curves were obtained by applying increasing concentrations of the agonists BK and DBK. These effects were blocked by the antagonists Icatibant and R-715, respectively. The potency (pD(2)) calculated from the curves was 7.0 +/- 0.1 for BK and 7.3 +/- 0.2 for DBK. The efficacy was 51 +/- 2% for BK and 30 +/- 1% for DBK when compared to 1 microM norepinephrine. The concentration-dependent responses of BK and DBK were markedly inhibited by the arachidonic acid inhibitor indomethacin (1 microM), suggesting a mediation by the cyclooxygenase pathway. These contractile responses were not potentiated in the presence of the NOS inhibitor L-NAME (1 mM) or endothelium-denuded aorta, indicating that the NO pathway is not involved. We conclude that the mouse aorta constitutively contains B(1) and B(2) subtypes of kinin receptors and that stimulation with BK and DBK induces contractile effect mediated by endothelium-independent vasoconstrictor prostanoids.

The role of the renal kallikrein-kinin system in diabetic nephropathy.

PURPOSE OF REVIEW: Diabetic nephropathy is one of the most common complications in diabetes mellitus. Multiple pathogenic mechanisms are now believed to contribute to this disease, including inflammatory cytokines, autacoids and oxidative stress. Numerous studies have shown that the kallikrein-kinin system may be involved in these mechanisms. This review focuses on recent research advance on the potential role of the kallikrein-kinin system in the development of diabetic nephropathy, and its clinical relevance. RECENT FINDINGS: A collection of recent studies has shown that angiotensin-converting enzyme inhibitors, which inhibit angiotensin II formation and degradation of bradykinin, and vasopeptidase inhibitors attenuated the development of diabetic nephropathy in experimental animals and clinical settings. The role of the kallikrein-kinin system in diabetes is further supported by findings that diabetic nephropathy is worsened in diabetic mice lacking bradykinin B2 receptors. Although long-acting bradykinin B2 receptor agonists have been shown to have renal protective effects, their therapeutic benefits have not been well studied. SUMMARY: Current experimental investigations demonstrated that pharmacological intervention of the kallikrein-kinin system improved renal conditions in diabetes mellitus. These findings suggest that the kallikrein-kinin system may be a therapeutic target in preventing and treating diabetic nephropathy.

Systemic inhibition of the angiotensin-converting enzyme limits lipopolysaccharide-induced lung neutrophil recruitment through both bradykinin and angiotensin II-regulated pathways.

Recruitment of neutrophils to the lung is a sentinel event in acute lung inflammation. Identifying mechanisms that regulate neutrophil recruitment to the lung may result in strategies to limit lung damage and improve clinical outcomes. Recently, the renin angiotensin system (RAS) has been shown to regulate neutrophil influx in acute inflammatory models of cardiac, neurologic, and gastrointestinal disease. As a role for the RAS in LPS-induced acute lung inflammation has not been described, we undertook this study to examine the possibility that the RAS regulates neutrophil recruitment to the lung after LPS exposure. Pretreatment of mice with the angiotensin-converting enzyme (ACE) inhibitor enalapril, but not the anti-hypertensive hydralazine, decreased pulmonary neutrophil recruitment after exposure to LPS. We hypothesize that inhibition of LPS-induced neutrophil accumulation to the lung with enalapril occurred through both an increase in bradykinin, and a decrease in angiotensin II (ATII), mediated signaling. Bradykinin receptor blockade reversed the inhibitory effect of enalapril on neutrophil recruitment. Similarly, pretreatment with bradykinin receptor agonists inhibited IL-8-induced neutrophil chemotaxis and LPS-induced neutrophil recruitment to the lung. Inhibition of ATII-mediated signaling, with the ATII receptor 1a inhibitor losartan, decreased LPS-induced pulmonary neutrophil recruitment, and this was suggested to occur through decreased PAI-1 levels. LPS-induced PAI-1 levels were diminished in animals pretreated with losartan and in those deficient for the ATII receptor 1a. Taken together, these results suggest that ACE regulates LPS-induced pulmonary neutrophil recruitment via modulation of both bradykinin- and ATII-mediated pathways, each regulating neutrophil recruitment by separate, but distinct, mechanisms.

Hemorphin and hemorphin-like peptides isolated from dog pancreas and sheep brain are able to potentiate bradykinin activity in vivo.

Hemorphins are biologically active peptides, derived from hemoglobin, which presents a number of physiological activities. Proteolytic generation of these peptides is not fully understood; however, among their roles, is to provoke reduction on blood pressure. In this work, this particular biological effect was chosen as the monitor for the selection of mammalian vasoactive peptides. By combining high-performance liquid chromatography and mass spectrometry, including 'de novo' sequencing, several hemorphin-like peptides were identified presenting bradykinin potentiating activity. Moreover, taking LVV-hemorphin-7 as model compound, we evaluated its biological effect on blood pressure of anaesthetized rats. By summarizing all the results, it is possible to present the hemorphins as a family of proteolytically generated peptides that are able to potentiate bradykinin activity in vivo.

Small peptides, big world: biotechnological potential in neglected bioactive peptides from arthropod venoms.

Until recently, a toxinologist's tasks involved the search for highly toxic or lethal toxins in animal venoms that could explain the harmful effects in clinically observed symptoms. Most of these toxins were put on evidence using a function to structure approach, in which a biological phenomena observation usually guided the isolation and characterization of the causative molecule. Paving this way, many toxins were promptly purified because of their readily observed effect. Nevertheless, small molecules with micro-effects that are not easily visualized can be relatively neglected or poorly studied. This situation has changed now with the advent of the sensitivity, resolution and accuracy of techniques such as mass spectrometry and proteomic approaches used in toxinology. Taking advantage of these methodologies, small peptides with 'newly exploited' biological activities such as vasoactive, hormone-like, antimicrobial and others have been recently given much more attention, enlarging the known repertoire of bioactive molecules found in animal venoms. This article aims to review current knowledge on small biologically active peptides (<3 kDa) found in arthropod venoms and discuss their potentialities as new drug candidates or therapeutic lead compounds.

Hypotensive agents from snake venoms.

Many snake venoms contain toxins which produce profound cardiovascular effects. The site of action of these toxins includes cardiac muscle, vascular smooth muscle and the capillary vascular bed. Some snake venoms, for example, contain peptides that inhibit angiotensin converting enzyme and potentiate the biological actions of bradykinin. Other snake venoms contain structural and functional equivalents of mammalian natriuretic peptides. Sarafotoxins are short peptide toxins found in the venoms of snakes from Atractaspis spp. which display potent vasoconstriction properties. These peptides, which share a high degree of sequence identity with endothelins, recognize and bind to endothelin receptors. Snakes have also evolved toxins which block L-type Ca(2+) currents (eg. calciseptine, FS2 toxins, C(10)S(2)C(2) and S(4)C(8)). Snake venom proteins have also been shown to increase vascular permeability. One such protein, increasing capillary permeability protein (ICPP) has recently been isolated from the venom of Vipera lebetina. ICPP is an extremely potent permeability factor with a structure similar to vascular endothelial growth factor (VEGF). Thus there is a vast array of snake toxins with potent cardiovascular activity. Some of these proteins and peptides have proven to be highly selective tools in the study of physiological processes. Others have been used as probes of potential therapeutic targets or as lead compounds in the development of therapeutic agents. Therefore these and other related snake venom proteins hold great promise in the future understanding and treatment of cardiovascular diseases.

BK2 but not BK1 receptors mediating contractile response in human umbilical arteries: role of thromboxane A2.

Bradykinin receptors have been divided into B1 and B2 subtypes. The aim of this study on human umbilical arteries was: i) to compare the recognition properties of the mediating contractions of bradykinin receptors; and ii) to assess the possible role of thromboxane A2 in a bradykinin-induced contraction of smooth muscle. Umbilical arteries were dissected and mounted in organ baths for isometric measurement of force. Our results showed that the B1 agonist [Sar1dPhe8desArg9]-bradykinin had no effect on the concentration-response 10(-9)-3 x 10(-5) mM. Cumulative additions of bradykinin (10(-9)-3 x 10(-5) mM) and of the B2 agonist [Hyp3TyrMe8]-bradykinin (10(-9)-3 x 10(-5) mM) produced dose-dependent contractions. Dose response curves to bradykinin (10(-9)-3 x 10(-5) mM) were not significantly altered by the presence of B1 selective antagonist [des-Arg9, Leu8]-bradykinin (10(-5) mM), or by the selective B2 antagonist [Thi(5,8), D-Phe7]-bradykinin (10(-5) mM). However, Hoe 140 D-Arg-[Hyp3, Thi5,D-Tic7, Oic8]-bradykinin, an antagonist of B2 responses, significantly inhibited bradykinin-induced contraction. The responses to bradykinin were unaffected by indomethacin (10(-4) mM), dazoxiben (10(-5) mM) or even by nordihydroguaiaretic acid (10(-5) mM). However, bradykinin contractions were antagonized in a noncompetitive manner by quinacrine (10(-5) mM). These results showed that bradykinin contracts human umbilical arteries essentially through B2 receptors. Moreover, the responses to bradykinin are unlikely to be mediated by the cyclooxygenase/lipooxygenase pathway. The inhibitory effects of quinacrine may be due to a specific or nonspecific effect at a cellular level on smooth muscle contractility, or due to a direct action to block Ca2+ influx at membrane level.

Bradykinin, angiotensin-(1-7), and ACE inhibitors: how do they interact?

The beneficial effect of ACE inhibitors in hypertension and heart failure may relate, at least in part, to their capacity to interfere with bradykinin metabolism. In addition, recent studies have provided evidence for bradykinin-potentiating effects of ACE inhibitors that are independent of bradykinin hydrolysis, i.e. ACE-bradykinin type 2 (B(2)) receptor 'cross-talk', resulting in B(2) receptor upregulation and/or more efficient activation of signal transduction pathways, as well as direct activation of bradykinin type 1 receptors by ACE inhibitors. This review critically reviews the current evidence for hydrolysis-independent bradykinin potentiation by ACE inhibitors, evaluating not only the many studies that have been performed with ACE-resistant bradykinin analogues, but also paying attention to angiotensin-(1-7), a metabolite of both angiotensin I and II, that could act as an endogenous ACE inhibitor. The levels of angiotensin-(1-7) are increased during ACE inhibition, and most studies suggest that its hypotensive effects are mediated in a bradykinin-dependent manner.

Activation of phospholipase D by bradykinin and sphingosine 1-phosphate in A549 human lung adenocarcinoma cells via different GTP-binding proteins and protein kinase C delta signaling pathways.

Phospholipase D (PLD) is involved in the signaling by many extracellular ligands, and its regulation appears to be quite complex. We investigated the signaling pathways initiated by bradykinin (BK) or sphingosine 1-phosphate (S1P) in A549 cells to define molecular mechanisms responsible for their additive effects on PLD activity. BK and S1P each elicited a sustained increase in phosphatidic acid content through a rapid and transient activation of PLD. The two pathways demonstrated rapid homologous downregulation, but heterologous desensitization was not observed. Action of both agonists required protein kinase C (PKC) activation and Ca(2+) influx but was mediated by different heterotrimeric G proteins. In membranes, inhibition of PKCdelta by rottlerin enhanced BK activation of PLD but inhibited that by S1P. Rottlerin inhibited activation of PLD in nuclei by both BK and S1P. By in situ immunofluorescence or cell fractionation followed by immunoblotting, PLD1 was concentrated primarily in nuclei, whereas the membrane fraction contained PLD2 and PLD1. Moreover, PKCdelta specifically phosphorylated recombinant PLD2, but not PLD1. BK and S1P similarly enhanced RhoA translocation to nuclei, whereas BK was less efficacious than S1P on RhoA relocalization to membranes. Effects of both agonists on the nuclear fraction, which contains only PLD1, are compatible with a RhoA- and PKCdelta-dependent process. In membranes, which contain both PLD1 and PLD2, the stimulatory effect of S1P on PLD activity can best be explained by RhoA- and PKCdelta-dependent activation of PLD1; in contrast, the effects of BK on RhoA translocation and enhancement of BK-stimulated PLD activity by PKC inhibition are both consistent with PLD2 serving as its primary target.

Mechanisms underlying the contraction induced by bradykinin in the guinea pig epithelium-denuded trachea.

This study investigates some of the mechanisms by which bradykinin (BK) triggers contraction of epithelium-denuded strips of guinea pig trachea (GPT). Cumulative or single additions of BK, T-BK, L-BK, or ML-BK in the presence of captopril (30 microM) produced graded GPT contractions with the following rank order of potency (EC50 level): T-BK (31.3 nM) > BK (40.0 nM) > L-BK (56.0 nM) > ML-BK (77.0 nM). BK-induced contraction (100 nM) in GPT was completely inhibited by either HOE 140 or NPC 17731 with mean IC50 values of 17 and 217 nM, respectively. Addition of BK (100 nM) at 30 min intervals, induced progressive tachyphylaxis, which was complete after 4 h. The tachyphylaxis induced by BK was unaffected by L-NOARG (nitric oxide synthase inhibitor, 100 microM) or valeryl salicylate (a cyclooxygenase-1 (COX-1) inhibitor, 30 microM), but was prevented by a low concentration of indomethacin, diclofenac (non-selective COX inhibitors, 3 nM each) or by NS 398 (a COX-2 inhibitor, 10 nM). Furthermore, higher concentrations of indomethacin, diclofenac, phenidone (a lypooxygenase (LOX) and COX inhibitor), or NS 398, caused graded inhibition of BK-induced contraction, with mean IC50 values of 0.28, 0.08, 46.37, and 0.15 microM, respectively. Together, these results suggest that BK-induced contraction in GPT involves activation of B2 receptors and release of prostanoids from COX-2 pathway. Furthermore, the tachyphylaxis induced by BK was insensitive to the nitric oxide and COX-1 inhibitors, but was prevented by non-selective and selective COX-2 inhibitors, indicating a mediation via COX-2-derived arachidonic acid metabolites.

Selective inhibition of the C-domain of angiotensin I converting enzyme by bradykinin potentiating peptides.

Somatic angiotensin I converting enzyme (ACE) contains two functional active sites. Up to now, most of the studies aimed at characterizing the selectivity of inhibitors toward the two ACE active sites relied on the use of ACE mutants containing a single functional active site. By developing new fluorogenic synthetic substrates of ACE, we demonstrated that inhibitor selectivity can be assessed directly by using somatic ACE. This useful screening approach led us to discover that some bradykinin potentiating peptides turned out to be selective inhibitors of the C-domain of ACE. The peptide pGlu-Gly-Leu-Pro-Pro-Arg-Pro-Lys-Ile-Pro-Pro, with K(i)(app) values of 30 nM and 8 microM, respectively, for the C- and N-domain of ACE, is to our knowledge the most highly selective C-domain inhibitor of ACE so far reported. Inhibitors able to block selectively either the N- or C-domain of ACE will represent unique tools to probe the function of each domain in the regulation of blood pressure or other physiopathological events involving ACE activity.

No evidence for bradykinin B1 receptors in rat dorsal root ganglion neurons.

Bradykinin receptors are believed to contribute to hyperalgesia under conditions of neuropathic pain. Using calcium imaging we investigated responses to B1 and B2 agonists on isolated rat dorsal root ganglion neurons. No response to the B1 agonist was detected, whereas 12% of neurons responded to the B2 agonist. Northern blot analysis confirmed the lack of B1 receptor expression in dorsal root ganglia, as B1 mRNA was neither detected under normal conditions nor after nerve injury. In the calcium imaging experiments, agonists were applied with an elevated superfusion flow rate to avoid tachyphylaxis to the drug. Normal external solution applied at this flow rate constituted a mechanical stimulus causing a response in some neurons. Thus, in comparable set-ups mechanosensitivity has first to be tested to avoid masking effects.